Method of expressing Heliothis ecdysone receptor fusion protein

ABSTRACT

The invention relates to an insect steroid receptor protein which is capable of acting as a gene switch which is responsive to a chemical inducer enabling external control of the gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 09/564,418,filed May 3, 2000, now U.S. Pat. No. 6,610,828 which is a divisional ofU.S. patent application Ser. No. 08/653,648, filed May 24, 1996, nowU.S. Pat. No. 6,379,945. The entire contents of U.S. patent applicationSer. Nos. 08/653,648 and 09/564,418 are herein incorporated byreference.

TECHNICAL FIELD

The present invention relates to the identification and characterisationof insect steroid receptors from the Lepidoptera species Heliothisvirescens, and the nucleic acid encoding therefor. The present inventionalso relates to the use of such receptors, and such nucleic acid,particularly, but not exclusively, in screening methods, and geneswitches. By gene switch we mean a gene sequence which is responsive toan applied exogenous chemical inducer enabling external control ofexpression of the gene controlled by said gene sequence.

BACKGROUND ART

Lipophilic hormones such as steroids induce changes in gene expressionto elicit profound effects on growth, cellular differentiation, andhomeostasis. These hormones recognise intracellular receptors that sharea common modular structure consisting of three main functional domains:a variable amino terminal region that contains a transactivation domain,a DNA binding domain, and a ligand binding domain on the carboxyl sideof the molecule. The DNA binding domain contains nine invariantcysteines, eight of which are involved in zinc coordination to form atwo-finger structure. In the nucleus the hormone-receptor complex bindsto specific enhancer-like sequences called hormone response elements(HREs) to modulate transcription of target genes.

The field of insect steroid research has undergone a revolution in thelast three years as a result of the cloning and preliminarycharacterisation of the first steroid receptor member genes. Thesedevelopments suggest the time is ripe to try to use this knowledge toimprove our tools in the constant fight against insect pests. Most ofthe research carried out on the molecular biology of the steroidreceptor superfamily has been on Drosophila melanogaster (Diptera), seefor example International Patent Publication No. WO 91/13167, with somein Manduca and Galleria (Lepidoptera).

It has been three decades since 20-hydroxyecdysone was first isolatedand shown to be involved in the regulation of development of insects.Since then work has been carried out to try to understand the pathway bywhich this small hydrophobic molecule regulates a number of activities.By the early 1970s, through the studies of Clever and Ashburner, it wasclear that at least in the salivary glands of third instar Drosophilalarvae, the application of ecdysone lead to the reproducible activationof over a hundred genes. The ecdysone receptor in this pathway isinvolved in the regulation of two classes of genes: a small class (earlygenes) which are induced by the ecdysone receptor and a large class(late genes) which are repressed by the ecdysone receptor. The earlyclass of genes are thought to have two functions reciprocal to those ofthe ecdysone receptor; the repression of the early transcripts and theinduction of late gene transcription. Members of the early genes so farisolated and characterised belong to the class of molecules withcharacteristics similar to known transcription factors. They are thuspredicted to behave as expected by the model of ecdysone action(Ashburner, 1991). More recently, the early genes E74 and E75 have beenshown to bind both types of ecdysone inducible genes (Thummel et al.,1990; Segraves and Hogness, 1991), thus supporting their proposed dualactivities. It should be noted however, that the activation of ahierarchy of genes is not limited to third instar larvae salivaryglands, but that the response to the ecdysone peak at the end of larvallife is observed in many other tissues, such as the imaginal disks (i.e.those tissues which metamorphose to adult structures) and other larvaltissues which histolyse at the end of larval life (e.g. larval fatbody). The model for ecdysone action as deduced by studying the thirdinstar chromosome puffing may not apply to the activation of ecdysoneregulated genes in adults. In other words, the requirement for otherfactors in addition to the active ecdysone receptor must be satisfiedfor correct developmental expression (e.g. the Drosophila yolk proteingene expression in adults is under control of doublesex, the last genein the sex determination gene hierarchy).

The ecdysone receptor and the early gene E75 belong to the steroidreceptor superfamily. Other Drosophila genes, including ultraspiracle,tailless, sevenup and FTZ-FI, also belong to this family. However, ofall these genes only the ecdysone receptor is known to have a ligand,and thus the others are known as orphan receptors. Interestingly,despite the ultraspiracle protein ligand binding region sharing 49%identity with the vertebrate retinoic X receptor (RXR) ligand bindingregion (Oro et al., 1990), they do not share the same ligand (i.e. theRXR ligand is 9-cis retinoic acid) (Heymann et al., 1992 and Mangelsdorfet al., 1992). All the Drosophila genes mentioned are involved indevelopment, ultraspiracle for example, is required for embryonic andlarval abdominal development. The protein products of these genes allfit the main features of the steroid receptor superfamily (Evans, 1988;Green and Chambon, 1988, Beato, 1989): i.e., they have a variable Nterminus region involved in ligand independent transactivation (DomainsA and B), a highly conserved 66–68 amino acid region which isresponsible for the binding of DNA at specific sites (Domain C), a hingeregion thought to contain a nuclear translocation signal (Domain D), anda well conserved region containing the ligand binding region,transactivation sequences and the dimerisation phase (Domain E). Thelast region, domain F, is also very variable and its function isunknown.

Steroid receptor action has been elucidated in considerable detail invertebrate systems at both the cellular and molecular levels. In theabsence of ligand, the receptor molecule resides in the cytoplasm whereit is bound by Hsp90, Hsp70, and p59 to form the inactive complex(Evans, 1988). Upon binding of the ligand molecule by the receptor aconformational change takes place which releases the Hsp90, Hsp70 andp59 molecules, while exposing the nuclear translocation signals in thereceptor. The ligand dependent conformational change is seen in theligand binding domain of both progesterone and retinoic acid receptors(Allan et al., 1992a). This conformational change has been furthercharacterised in the progesterone receptor and was found to beindispensable for gene transactivation (Allan et al., 1992b). Onceinside the nucleus the receptor dimer binds to the receptor responsiveelement at a specific site on the DNA resulting in the activation orrepression of a target gene. The receptor responsive elements usuallyconsist of degenerate direct repeats, with a spacer between 1 and 5nucleotides, which are bound by a receptor dimer through the DNA bindingregion (Domain C).

Whereas some steroid hormone receptors are active as homodimers othersact as heterodimers. For example, in vertebrates, the retinoic acidreceptor (RAR) forms heterodimers with the retinoic X receptor (RXR).RXR can also form heterodimers with the thyroid receptor, vitamin Dreceptor (Yu et al., 1991; Leid et al., 1992) and peroxisome activatorreceptor (Kliewer et al., 1992). Functionally the main differencebetween homodimers and heterodimers is increased specificity of bindingto specific response elements. This indicates that different pathwayscan be linked, co-ordinated and modulated, and more importantly thisobservation begins to explain the molecular basis of the pleotropicactivity of retinoic acid in vertebrate development (Leid et al.,1992b). Similarly, the Drosophila ultraspiracle gene product wasrecently shown to be capable of forming heterodimers with retinoic acid,thyroid, vitamin D and peroxisome activator receptors and to stimulatethe binding of these receptors to their target responsive elements (Yaoet al., 1993). More significantly, the ultraspiracle gene product hasalso been shown to form heterodimers with the ecdysone receptor,resulting in cooperative binding to the ecdysone response element andcapable of rendering mammalian cells ecdysone responsive (Yao et al.,1992). The latter is of importance since transactivation of the ecdysonegene alone in mammalian cells fails to elicit an ecdysone response(Koelle et al., 1991), therefore suggesting that the ultraspiracle geneproduct is an integral component of a functional ecdysone receptor (Yaoet al., 1992). It is possible that the ultraspiracle product competeswith other steroid receptors or factors to form heterodimers with theecdysone receptor. Moreover it remains to be investigated ifultraspiracle is expressed in all tissues of the Drosophila larvae.Despite ultraspiracle being necessary to produce a functional ecdysonereceptor, the mechanism by which this activation takes place is as yetundetermined.

SUMMARY

We have now isolated and characterised the ecdysone steroid receptorfrom Heliothis virescens (hereinafter HEcR). We have found thatsurprisingly unlike the Drosophila ecdysone steroid receptor(hereinafter DEcR) in reports to-date, HEcR can be induced by knownnon-steroidal inducers. It will be appreciated that this provides manyadvantages for the system.

Steroids are difficult and expensive to make. In addition, the use of anon-steroid as the inducer allows the system to be used in agrochemicaland pharmaceutical applications, not least because it avoids applicationof a steroid which is already present in insects and/or mammals. Forexample, it would not be feasible to use a gene switch in a mammaliancell which was induced by a naturally occurring steroidal inducer. Itwill also be appreciated that for environmental reasons it isadvantageous to avoid the use of steroids as inducers.

According to one aspect of the present invention there is provided DNAhaving the sequence shown in SEQ ID NO: 2, wherein SEQ ID NO: 2 givesthe sequence for the HEcR.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 2, which encodes forthe HEcR ligand binding domain.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 2, which encodes forthe HEcR DNA binding domain.

According to yet another aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 2, whichencodes for the HEcR transactivation domain.

According to a further aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 2, which encodes forthe HEcR hinge domain.

According to a still further aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 2, whichencodes for the HEcR carboxy terminal region.

According to one aspect of the present invention there is provided DNAhaving the sequence shown in SEQ ID NO: 3, wherein SEQ ID NO: 3 givesthe sequence for the HEcR.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 3, which encodes forthe HEcR ligand binding domain.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 3, which encodes forthe HEcR DNA binding domain.

According to yet another aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 3, whichencodes for the HEcR transactivation domain.

According to a further aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 3, which encodes forthe HEcR hinge domain.

According to a still further aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 3, whichencodes for the HEcR carboxy terminal region.

According to one aspect of the present invention there is provided DNAhaving the sequence shown in SEQ ID NO: 4, wherein SEQ ID NO: 4 givesthe sequence for the HEcR.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 4, which encodes forthe HEcR ligand binding domain.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 4, which encodes forthe HEcR DNA binding domain.

According to yet another aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 4, whichencodes for the HEcR transactivation domain.

According to a further aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 4, which encodes forthe HEcR hinge domain.

According to a still further aspect of the present invention there isprovided DNA having part of the sequence shown in SEQ ID NO: 4, whichencodes for the HEcR carboxy terminal region.

DETAILED DESCRIPTION

As mentioned above, steroid receptors are eukaryotic transcriptionalregulatory factors which, in response to the binding of the steroidhormone, are believed to bind to specific DNA elements and activatetranscription. The steroid receptor can be divided into six regions,designated A to F, using alignment techniques based on shared homologywith other members of the steroid hormone receptor superfamily. Krust etal. (1986) identified two main regions in the receptor, C and E. RegionC is hydrophilic and is unusual in its high content in cysteine, lysineand arginine. It corresponds to a DNA-binding domain, sometimes referredto as the “zinc finger”. It is the DNA binding domain which binds to theupstream DNA of the responsive gene. Such upstream DNA is known as thehormone response element or HRE for short. Region E is hydrophobic andis identified as the hormone (or ligand) binding domain. Region E can befurther subdivided into regions E1, E2 and E3.

The region D, which separates domains C and E is highly hydrophobic andis flexible. It is believe that communication between domains E and Cinvolves direct contact between them through region D, which provides ahinge between the two domains. Region D is therefore referred to as thehinge domain.

The mechanism of the receptor appears to require it to interact withsome element(s) of the transcription machinery over and above itsinteractions with the hormone and the hormone response element.N-terminal regions A and B perform such a function and are jointly knownas the transactivation domain. The carboxy terminal region is designatedF.

The domain boundaries of the HEcR can be defined as follows:

INTERVALS DOMAIN base pairs amino acids Transactivating (A/B) 114–600 1–162 DNA Binding (C) 601–798 163–228 Hinge (D)  799–1091 229–326Ligand Binding (E) 1092–1757 327–545 C-Terminal End (F) 1758–1844546–577

The DNA binding domain is very well defined and is 66 amino acids long,thus providing good boundaries. The above intervals have been definedusing the multiple alignment for the ecdysone receptors (FIG. 5).

The present invention also includes DNA which shows homology to thesequences of the present invention. Typically homology is shown when 60%or more of the nucleotides are common, more typically 65%, preferably70%, more preferably 75%, even more preferably 80% or 85%, especiallypreferred are 90%, 95%, 98% or 99% or more homology.

The present invention also includes DNA which hybridises to the DNA ofthe present invention and which codes for at least part of the Heliothisecdysone receptor transactivation domain, DNA binding domain, hingedomain, ligand binding domain and/or carboxy terminal region. Preferablysuch hybridisation occurs at, or between, low and high stringencyconditions. In general terms, low stringency conditions can be definedas 3×SSC at about ambient temperature to about 65° C., and highstringency conditions as 1.0×SSC at about 65° C. SSC is the name of abuffer of 0.15M NaCl, 0.015M trisodium citrate. 3×SSC is three times asstrong as SSC and so on.

The present invention further includes DNA which is degenerate as aresult of the genetic code to the DNA of the present invention and whichcodes for a polypeptide which is at least part of the Heliothis ecdysonereceptor transactivation domain, DNA binding domain, hinge domain,ligand binding domain and/or carboxy terminal region.

The DNA of the present invention may be cDNA or DNA which is in anisolated form.

According to another aspect of the present invention there is provided apolypeptide comprising the Heliothis ecdysone receptor or a fragmentthereof, wherein said polypeptide is substantially free from otherproteins with which it is ordinarily associated, and which is coded forby any of the DNA of the present invention.

According to another aspect of the present invention there is provided apolypeptide which has the amino acid sequence of SEQ ID NO: 5 or anyallelic variant or derivative thereof, wherein SEQ ID NO: 5 gives theamino acid sequence of the HEcR polypeptide.

According to another aspect of the present invention there is provided apolypeptide which has part of the amino acid sequence of SEQ ID NO: 5 orany allelic variant or derivative thereof, which sequence provides theHEcR ligand binding domain.

According to another aspect of the present invention there is provided apolypeptide which has part of the amino acid sequence of SEQ ID NO: 5 orany allelic variant or derivative thereof, which sequence provides theHEcR DNA binding domain.

According to yet another aspect of the present invention there isprovided a polypeptide which has part of the amino acid sequence of SEQID NO: 5 or any allelic variant or derivative thereof, which sequenceprovides the HEcR transactivation domain.

According to a further aspect of the present invention there is provideda polypeptide which has the amino acid sequence of a part of SEQ ID NO:5 or any allelic variant or derivative thereof, which sequence providesthe HEcR hinge domain.

According to a still further aspect of the present invention there isprovided a polypeptide which has the amino acid sequence of a part ofSEQ ID NO: 5 or any allelic variant or derivative thereof, whichsequence provides the HEcR carboxy terminal region.

For the avoidance of doubt, spliced variants of the amino acid sequencesof the present invention are included in the present invention.

Preferably, said derivative is a homologous variant which hasconservative amino acid changes. By conservation amino acid changes wemean replacing an amino acid from one of the amino acid groups, namelyhydrophobic, polar, acidic or basic, with an amino acid from within thesame group. An example of such a change is the replacement of valine bymethionine and vice versa.

According to another aspect of the present invention there is provided afusion polypeptide comprising at least one of the polypeptides of thepresent invention functionally linked to an appropriate non-Heliothisecdysone receptor domain(s).

According to an especially preferred embodiment of the present inventionthe HEcR ligand binding domain of the present invention is fused to aDNA binding domain and a transactivation domain.

According to another embodiment of the present invention the DNA bindingdomain is fused to a ligand binding domain and a transactivation domain.

According to yet another embodiment of the present invention thetransactivation domain is fused to a ligand binding domain and a DNAbinding domain.

The present invention also provides recombinant DNA encoding for thesefused polypeptides.

According to an especially preferred embodiment of the present inventionthere is provided recombinant nucleic acid comprising a DNA sequenceencoding the HEcR ligand binding domain functionally linked to DNAencoding the DNA binding domain and transactivation domain from aglucocorticoid receptor.

According to yet another aspect of the present invention there isprovided recombinant nucleic acid comprising a DNA sequence comprising areporter gene operably linked to a promoter sequence and a hormoneresponse element which hormone response element is responsive to the DNAbonding domain encoded by the DNA of the present invention.

According to another aspect of the present invention there is provided aconstruct transformed with nucleic acid, recombinant DNA, a polypeptideor a fusion polypeptide of the present invention. Such constructsinclude plasmids and phages suitable for transforming a cell ofinterest. Such constructs will be well known to those skilled in theart.

According to another aspect of the present invention there is provided acell transformed with nucleic acid, recombinant DNA, a polypeptide, or afusion polypeptide of the present invention.

Preferably the cell is a plant, fungus or mammalian cell.

For the avoidance of doubt fungus includes yeast.

The present invention therefore provides a gene switch which is operablylinked to a foreign gene or a series of foreign genes whereby expressionof said foreign gene or said series of foreign genes may be controlledby application of an effective exogenous inducer.

Analogs of ecdysone, such as Muristerone A, are found in plants anddisrupt the development of insects. It is therefore proposed that thereceptor of the present invention can be used be in plants transformedtherewith as an insect control mechanism. The production of theinsect-damaging product being controlled by an exogenous inducer. Theinsect-damaging product can be ecdysone or another suitable protein.

The first non-steroidal ecdysteroid agonists, dibenzoyl hydrazines,typified by RH-5849 [1,2-dibenzoyl, 1-tert-butyl hydrazide], which iscommercially available as an insecticide from Rohm and Haas, weredescribed back in 1988. Another commercially available compound in thisseries is RH-5992 [tebufenozide, 3,5-dimethylbenzoic acid1-1(1,1-dimethylethyl)-2(4-ethylbenzoyl)hydrazide]. These compoundsmimic 20-hydroxyecdysone (20E) in both Manduca sexta and Drosophilamelanogaster. These compounds have the advantage that they have thepotential to control insects using ecdysteroid agonists which arenon-steroidal. Further Examples of such dibenzoyl hydrazines are givenin U.S. Pat. No. 5,117,057 to Rohm and Haas, and Oikawa et al., PesticSci, 41, 139–148 (1994). However, it will be appreciated that anyinducer of the gene switch of the present invention, whether steroidalor non-steroidal, and which is currently or becomes available, may beused.

The gene switch of the present invention, then, when linked to anexogenous or foreign gene and introduced into a plant by transformation,provides a means for the external regulation of expression of thatforeign gene. The method employed for transformation of the plant cellsis not especially germane to this invention and any method suitable forthe target plant may be employed. Transgenic plants are obtained byregeneration from the transformed cells. Numerous transformationprocedures are known from the literature such as agroinfection usingAgrobacterium tumefaciens or its Ti plasmid, electroporation,microinjection or plants cells and protoplasts, microprojectiletransformation, to mention but a few. Reference may be made to theliterature for full details of the known methods.

Neither is the plant species into which the chemically induciblesequence is inserted particularly germane to the invention.Dicotyledonous and monocotyledonous plants can be transformed. Thisinvention may be applied to any plant for which transformationtechniques are, or become, available. The present invention cantherefore be used to control gene expression in a variety of geneticallymodified plants, including field crops such as canola, sunflower,tobacco, sugarbeet, and cotton; cereals such as wheat, barley, rice,maize, and sorghum; fruit such as tomatoes, mangoes, peaches, apples,pears, strawberries, bananas and melons; and vegetables such as carrot,lettuce, cabbage and onion. The switch is also suitable for use in avariety of tissues, including roots, leaves, stems and reproductivetissues.

In a particularly preferred embodiment of the present invention, thegene switch of the present invention is used to control expression ofgenes which confer resistance herbicide resistance and/or insecttolerance to plants.

Recent advances in plant biotechnology have resulted in the generationof transgenic plants resistant to herbicide application, and transgenicplants resistant to insects. Herbicide tolerance has been achieved usinga range of different transgenic strategies. One well documented examplein the herbicide field is the use the bacterial xenobiotic detoxifyinggene phosphinothricin acetyl transferase (PAT) from Streptomyceshydroscopicus. Mutated genes of plant origin, for example the alteredtarget site gene encoding acetolactate synthase (ALS) from Arabidopsis,have been successfully utilised to generate transgenic plants resistantto herbicide application. The PAT and ALS genes have been expressedunder the control of strong constitutive promoter. In the field ofinsecticides, the most common example to-date is the use of the Bt gene.

We propose a system where genes conferring herbicide and/or insecttolerance would be expressed in an inducible manner dependent uponapplication of a specific activating chemical. This approach has anumber of benefits for the farmer, including the following:

-   1. Inducible control of herbicide and/or insect tolerance would    alleviate any risk of yield penalties associated with high levels of    constitutive expression of herbicide and/or insect resistance genes.    This may be a particular problem as early stages of growth where    high levels of transgene product may directly interfere with normal    development. Alternatively high levels of expression of herbicide    and/or insect resistance genes may cause a metabolic drain for plant    resources.-   2. The expression of herbicide resistance genes in an inducible    manner allows the herbicide in question to be used to control    volunteers if the activating chemical is omitted during treatment.-   3. The use of an inducible promoter to drive herbicide and/or insect    resistance genes will reduce the risk of resistance becoming a major    problem. If resistance genes were passed onto weed species from    related crops, control could still be achieved with the herbicide in    the absence of inducing chemical. This would particularly be    relevant if the tolerance gene confirmed resistance to a total    vegetative control herbicide which would be used (with no inducing    chemical) prior to sowing the crop and potentially after the crop    has been harvested. For example, it can be envisaged that herbicide    resistance cereals, such as wheat, might outcross into the weed wild    oats, thus conferring herbicide resistance to this already    troublesome weed. A further example is that the inducible expression    of herbicide resistance in sugar beet will reduce the risk of wild    sugar beet becoming a problem. Similarly, in the field of insect    control, insect resistance may well become a problem if the    tolerance gene is constitutively expressed. The used of an inducible    promoter will allow a greater range of insect resistance control    mechanisms to be employed.

This strategy of inducible expression of herbicide resistance can beachieved with a pre-spray of chemical activator or in the case of slowacting herbicides, for example N-phosphonomethyl-glycine (commonly knownas glyphosate), the chemical inducer can be added as a tank mixsimultaneously with the herbicide. Similar strategies can be employedfor insect control.

This strategy can be adopted for any resistance conferringgene/corresponding herbicide combination, which is, or becomes,available. For example, the gene switch of the present invention can beused with:

-   1. Maize glutathione S-transferase (GST-27) gene (see our PCT    International Patent Publication No. WO 90/08826), which confers    resistance to chloroacetanilide herbicides such as acetochlor,    metolachlor and alachlor.-   2. Phosphinotricin acetyl transferase (PAT), which confers    resistance to the herbicide commonly known as glufosinate.-   3. Acetolactate synthase gene mutants from maize (see our    International Patent Publication No. WO 90/14000) and other genes,    which confer resistance to sulphonyl urea and imadazolinones.-   4. Genes which confer resistance to glyphosate. Such genes include    the glyphosate oxidoreductase (GOX) gene (see International Patent    Publication No. WO 92/00377); genes which encode for    5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSPS), including    Class I and Class II EPSPS, genes which encode for mutant EPSPS, and    genes which encode for EPSPS fusion peptides such as that comprised    of a chloroplast transit peptide and EPSPS (see for example EP 218    571, EP 293 358, WO 91/04323, WO 92/04449 and WO 92/06201); and    genes which are involved in the expression of CP Lyase.

Similarly, the strategy of inducible expression of insect resistance canbe adopted for any tolerance-conferring gene which is, or becomes,available.

The gene switch of the present invention can also be used to controlledexpression of foreign proteins in yeast and mammalian cells. Manyheterologous proteins for many applications are produced by expressionin genetically engineered bacteria, yeast cells and other eukaryoticcells such as mammalian cells.

As well as the obvious advantage in providing control over theexpression of foreign genes in such cells, the switch of the presentinvention provides a further advantage in yeasts and mammalian cellswhere accumulation of large quantities of an heterologous protein candamage the cells, or where the heterologous protein is damaging suchthat expression for short periods of time is required in order tomaintain the viability of the cells.

Such an inducible system also has applicability in gene therapy allowingthe timing of expression of the therapeutic gene to be controlled. Thepresent invention is therefore not only applicable to transformedmammalian cells but also to mammals per se.

A further advantage of the inducible system of the present invention inmammalian cells is that, because it is derived from a insect, there isless chance of it being effected by inducers which effect the naturalmammalian steroid receptors.

In another aspect of the present invention the gene switch is used toswitch on genes which produce potentially damaging or lethal proteins.Such a system can be employed in the treatment of cancer in which cellsare transformed with genes which express proteins which are lethal tothe cancer. The timing of the action of such proteins on the cancercells can be controlled using the switch of the present invention.

The gene switch of the present invention can also be used to switchgenes off as well as on. This is useful in disease models. In such amodel the cell is allowed to grow before a specific gene(s) is switchedoff using the present invention. Such a model facilitates the study ofthe effect of a specific gene(s).

Again the method for producing such transgenic cells is not particularlygermane to the present invention and any method suitable for the targetcell may be used; such methods are known in the art, including cellspecific transformation.

As previously mentioned, modulation of gene expression in the systemappears in response to the binding of the HEcR to a specific control, orregulatory, DNA element. A schematic representation of the HEcR geneswitch is shown in FIG. 6. For ease of reference, the schematicrepresentation only shows three main domains of the HEcR, namely thetransactivation domain, DNA binding domain and the ligand bindingdomain. Binding of a ligand to the ligand binding domain enables the DNAbinding domain to bind to the HRE resulting in expression (or indeedrepression) of a target gene.

The gene switch of the present invention can therefore be seen as havingtwo components: a first component comprising the HEcR and a secondcomponent comprising an appropriate HRE and the target gene. Inpractice, the switch may conveniently take the form of one or twosequences of DNA, at least part of the one sequence, or one sequence ofthe pair, encoding the HEcR protein. Alternatively, the nucleic acidencoding the HEcR can be replaced by the protein/polypeptide itself.

Not only does the switch of the present invention have two components,but also one or more of the domains of the receptor can be variedproducing a chimeric gene switch. The switch of the present invention isvery flexible and different combinations can be used in order to varythe result/to optimise the system. The only requirement in such chimericsystems is that the DNA binding domain should bind to the hormoneresponse element in order to produce the desired effect.

The glucocorticoid steroid receptor is well characterised and has beenfound to work well in plants. A further advantage of this receptor isthat it functions as a homodimer. This means that there is no need toexpress a second protein such as the ultraspiracle in order to produce afunctional receptor. The problem with the glucocorticoid steroidreceptor is that ligands used to activate it are not compatible withagronomic practice.

In a preferred aspect of the present invention the receptor comprisesglucocorticoid receptor DNA binding and transactivation domains with aHeliothis ligand binding domain according to the present invention. Theresponse unit preferably comprising the glucocorticoid hormone responseelement and the desired effect gene. In the Examples, for convenience,this effect gene took the form of a reporter gene. However, in non-testor non-screen situations the gene will be the gene which produces thedesired effect, for example produces the desired protein. This proteinmay be a natural or exogenous protein. It will be appreciated that thischimeric switch combines the best features of the glucocorticoid system,whilst overcoming the disadvantage of only being inducible by a steroid.

In another preferred embodiment, the Heliothis ligand binding domain ischanged, and preferably replaced with a non-Heliothis ecdysone receptorligand binding domain. For example, we have isolated suitable sequencesfrom Spodoptera exigua.

Thus, according to another aspect of the present invention there isprovided DNA having the sequence shown in SEQ ID NO: 6.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 6, which encodes forthe Spodoptera ecdysone ligand binding domain.

According to another aspect of the present invention there is providedDNA having part of the sequence shown in SEQ ID NO: 6, which encodes forthe Spodoptera ecdysone hinge domain.

The present invention also provides the polypeptides coded for by theabove DNA sequences of SEQ ID NO: 6.

A further advantage with such chimeric systems is that they allow you tochoose the promoter which is used to drive the effector gene accordingto the desired end result. For example, placing the foreign gene underthe control of a cell specific promoter can be particularly advantageousin circumstances where you wish to control not only the timing ofexpression, but also which cells expression occurs in. Such a doublecontrol can be particularly important in the areas of gene therapy andthe use of cytotoxic proteins.

Changing the promoter also enables gene expression to be up- ordown-regulated as desired.

Any convenient promoter can be used in the present invention, and manyare known in the art.

Any convenient transactivation domain may also be used. Thetransactivation domain VP16 is a strong activator from Genentech Inc.,and is commonly used when expressing glucocorticoid receptor in plants.Other transactivation domains derived for example from plants or yeastmay be employed.

In a preferred embodiment of the present invention, the DNA bindingdomain is the glucocorticoid DNA binding domain. This domain is commonlya human glucocorticoid receptor DNA binding domain. However, the domaincan be obtained from any other convenient source, for example, rats.

According to another aspect of the present invention there is provided amethod of selecting compounds capable of being bound to an insectsteroid receptor superfamily member comprising screening compounds forbinding to a polypeptide or fusion polypeptide of the present invention,and selecting said compounds exhibiting said binding.

According to another aspect of the present invention there is provided acompound selected using the method of the present invention.

According to another aspect of the present invention there is providedan agricultural or pharmaceutical composition comprising the compound ofthe present invention.

According to yet another aspect of the present invention there isprovided the use of the compound of the present invention as apesticide, pharmaceutical and/or inducer of the switch. It will beappreciated that such inducers may well be useful as insecticides inthemselves.

According to a further aspect of the present invention there is provideda method of producing a protein or peptide or polypeptide comprisingintroducing into a cell of the present invention, a compound which bindsto the ligand binding domain in said cell.

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example with reference to theaccompanying examples and figures, in which figures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA sequence amplified from first strand cDNA made frommRNA isolated from Heliothis virescens Fourth instar larvae (SEQ IDNO: 1) The underlined sequences refer to the position of the degenerateoligonucleotides. At the 5′ end the sequence matches that of theoligonucleotide while at the 3′ end 12 nucleotides of the originaloligonucleotide are observed;

FIG. 2 shows the DNA sequence contained within the clone pSK19R (SEQ IDNO: 2) isolated from a random primed cDNA Heliothis virescens library;Sequence is flanked by EcoRI sites;

FIG. 3 shows the DNA sequence contained within the clone pSK16.1 (SEQ IDNO: 3) isolated from a random primed cDNA Heliothis virescens library;

FIG. 4 presents the DNA sequence of 5′ RACE products (in bold) fused tosequence of clone pSK16.1. The ORF (open reading frame; SEQ ID NO: 4)giving rise to the Heliothis virescens ecdysone receptor proteinsequence (SEQ ID NO: 5) is shown under the corresponding DNA sequence;

FIG. 5 shows the protein sequence alignment of the ecdysone receptorsDmEcR (Drosophila melanogaster; SEQ ID NO: 8), CtEcR (Chironomustentans; SEQ ID NO: 9), BmEcR (Bombyx mori; SEQ ID NO: 10), MsEcR(Manduca sexta; SEQ ID NO: 11), AaEcR (Aedes aegypti; SEQ ID NO: 12) andHvEcR (Heliothis virescens; SEQ ID NO: 5). “*” indicates conserved aminoacid residue. “.” indicates a conservative amino acid exchange;

FIG. 6 shows a model of an embodiment of the glucocorticoid/Heliothisecdysone chimeric receptor useable as a gene switch;

FIG. 7 shows a plasmid map of the clone pcDNA319R. The three othermammalian expression vectors were constructed in the same way and looksimilar but for the size of the insert;

FIG. 8 shows a plasmid map of the reporter construct used to analyse theactivity of the Heliothis virescens ecdysone receptor;

FIG. 9 is a graph which shows the effect of Muristerone A and RH5992 inreporter activity in HEK293 cells co-transfected with pcDNA3H3 KHEcRalone (filled bars) or with αRXR (stripped bars);

FIG. 10 shows a plasmid map of the Maize expression vector containingthe Glucocorticoid receptor (HG1 or pMF6HG1PAT);

FIG. 11 shows a plasmid map of the maize expression vector containingthe chimeric glucocorticoid/Drosophila ecdysone receptor pMF6GREcRS;

FIG. 12 shows a plasmid map of the maize expression vector containingthe chimeric glucocorticoid/Heliothis ecdysone receptor pMF6GRHEcR;

FIG. 13 shows a plasmid map of the plant reporter Plasmid containing theglucocorticoid response elements fused to the −60 S35CaMV promoter fusedto GUS, p221.9GRE6;

FIG. 14 shows a plasmid map of the plant reporter plasmid containing theglucocorticoid response elements fused to the −46 S35CaMV promoter fusedto GUS, p221.10GRE6;

FIG. 15 shows a graph showing the effect of Muristerone A andDexamethasone in Maize AXB protoplasts transformed with pMF6HG1PAT (GR)and p221.9GRE6 (reporter);

FIG. 16 shows a graph showing the effect of Muristerone A andDexamethasone in Maize AXB protoplasts transformed with pMF6GREcRS(effector) and p221.9GRE6 (reporter);

FIG. 17 shows a graph showing the effect of Muristerone A andDexamethasone in Maize AXB protoplasts transformed with pMF6GRHEcR(effector) and p221.9GRE6 (reporter);

FIG. 18 shows a graph showing the effect of RH5849 in Maize AXBprotoplasts transformed with pMF6GREcRS (effector) and p221.9GRE6(reporter);

FIG. 19 shows a graph showing the effect of RH5992 in Maize AXBprotoplasts transformed with pMF6GREcRS (effector) and p221.9GRE6(reporter);

FIG. 20 shows a graph showing the effect of RH5992 in Maize AXBprotoplasts transformed with pMF6GRHEcR (effector) and p221.9GRE6(reporter);

FIG. 21 shows a graph which shows the dose response effect of RH5992 inMaize AXB protoplasts transformed with pMF6GRHEcR (effector) andp221.9GRE6 (reporter);

FIG. 22 shows a plasmid map of the tobacco expression vector containingthe chimeric glucocorticoid/Drosophila ecdysone receptor, pMF7GREcRS;

FIG. 23 shows a plasmid map of the tobacco expression vector containingthe chimeric glucocorticoid/Heliothis ecdysone receptor, pMF7GRHEcR;

FIG. 24 shows a graph which shows the effect of RH5992 in Tobaccomesophyll protoplasts transformed with pMF6GRHEcR (Effector) andp221.9GRE6 (reporter);

FIG. 25 shows a plasmid map of the mammalian expression vectorcontaining the chimeric glucocorticoid/Heliothis ecdysone receptor,pcDNA3GRHEcR;

FIG. 26 shows a plasmid map of the reporter plasmid pSWGRE4;

FIG. 27 shows a graph which shows a RH5992 dose response curve of CHOcells transfected with pcDNA3GRHEcR and pSWGRE4;

FIG. 28 shows a graph which shows the effect of Muristerone A and RH5992on HEK293 cells co-transfected with pcDNA3GRHEcR and pSWGRE4;

FIG. 29 shows a plasmid map of the binary vector ES1;

FIG. 30 shows a plasmid map of the binary vector ES2;

FIG. 31 shows a plasmid map of the binary vector ES3;

FIG. 32 shows a plasmid map of the binary vector ES4;

FIG. 33 shows a plasmid map of the effector construct TEV-B112 made toexpress the HEcR ligand binding domain in yeast;

FIG. 34 shows a plasmid map of the effector construct TEV8 made toexpress the HEcR ligand binding domain in yeast;

FIG. 35 shows a plasmid map of the effector construct TEVVP16-3 made toexpress the HEcR ligand binding domain in yeast;

FIG. 36 shows a plasmid map of the mammalian expression vectorcontaining the chimeric glucocorticoid VP16/Heliothis ecdysone receptor,pcDNA3GRVP16HEcR;

FIG. 37 shows a plasmid map of the maize expression vector containingthe chimeric glucocorticoid VP16/Heliothis ecdysone receptor,pMF6GRVP16HEcR;

FIG. 38 shows a plasmid map of the maize expression vector containingthe chimeric glucocorticoid VP16/Heliothis ecdysone receptor,pMF7GRVP16HEcR;

FIG. 39 shows a graph which shows the effect of RH5992 in Maize AXBprotoplasts transformed with pMF6GRVP16HEcR (effector) and p221.9GRE6(reporter);

FIG. 40 shows the DNA sequence of the hinge and ligand binding domainsof the Spodoptera exigua ecdysone receptor (SEQ ID NO: 6);

FIG. 41 shows the protein sequence alignment of the Heliothis 19R (SEQID NO: 13) and Spodoptera SEcR Taq clone hinge and ligand bindingdomains (SEQ ID NO: 7). “*” indicates conserved amino acid residue. “.”indicates a conservative amino acid exchange;

FIG. 42 shows a graph which shows the effect of RH5992 on Tobaccomesophyll protoplasts transformed with pMF7GRHEcR (effector) and eitherp221.9GRE6 (Horizontal strips) or p221.10GRE6 (vertical strips).

EXAMPLE I Cloning of the Heliothis Ecdysone Receptor

A. Probe Generation

The rational behind the generation of the probe to isolate Heliothishomologues to the steroid/thyroid receptor superfamily members was basedon comparing the sequences of developmentally regulated steroid/thyroidreceptor superfamily members. The sequences available showed a highlyconserved motif within the DNA binding domain of the RAR and THR(thyroid) receptors. The motifs were used to design degenerateoligonucleotides for PCR amplification of sequences derived from cDNAtemplate produced from tissue expected to express developmentallyregulated steroid/thyroid receptor superfamily members (i.e. larvaltissues).

The sense oligonucleotide is based on the peptide sequence CEGCKGFF (SEQID NO: 14) which at the DNA level yields an oligonucleotide withdegeneracy of 32 as shown below:

ZnFA5′ 5′ TGC GAG GGI TGC AAG GAI TTC TT 3′ (SEQ ID NO: 15)     T   A       T   A       T

The antisense oligonucleotide is based on the reverse complementnucleotide sequence derived from the peptide:

CQECRLKK (SEQ ID NO: 16)   S  Rfor which four sets of degenerate oligos were made. Namely:

ZnFA3′ 5′ TTC TTI AGI CGG CAC TCT TGG CA 3′ (SEQ ID NO: 17)     T           A   T   C   A ZnFB3′ 5′ TTC TTI AAI CGG CAC TCT TGG CA3′ (SEQ ID NO: 18)      T           A   T   C   A ZnFC3′ 5′ TTC TTI AGICTG CAC TCT TGG CA 3′ (SEQ ID NO: 19)      T           A   T   C   AZnFD3′ 5′ TTC TTI AAI CTG CAC TCT TGG CA 3′ (SEQ ID NO: 20)     T           A   T   C   A

The PCR amplification was carried out using a randomly primed cDNAlibrary made from mRNA isolated from 4th and 5th instar Heliothisvirescens larvae. The amplification was performed using 10⁸ plaqueforming units (pfu) in 50 mM KCl. 20 mM Tris HCl pH 8.4, 15 mM MgCl2,200 mM dNTPs (an equimolar mixture of dCTP, dATP, dGTP and dTTP), 100 ngof ZnFA5′ and ZnF3′ mixture. The conditions used in the reactionfollowed the hot start protocol whereby the reaction mixture was heatedto 94° C. for 5 minutes after which 1 U of Taq polymerase was added andthe reaction allowed to continue for 35 cycles of 93° C. for 50 seconds,40° C. for 1 minute and 73° C. for 1 minute 30 seconds. The PCR productswere fractionated on a 2% (w/v) agarose gel and the ragment migratingbetween 100 and 200 bp markers was isolated and subcloned into thevector pCRII (Invitrogen). The sequence of the insert was determinedusing Sequenase (USB).

The resulting sequence was translated and a database search carried out.The search recovered sequences matching to the DNA binding domain of theDrosophila ecdysone receptor, retinoic acid receptor and the thyroidreceptor. Thus, the sequence of the insert in this plasmid, designatedpCRIIZnf, is a Heliothis ecdysone cognate sequence (FIG. 1) and was usedto screen a cDNA library in other to isolate the complete open readingframe.

B. Library Screening

The randomly primed cDNA 4th/5th Instar Heliothis virescens library wasplated and replicate filter made from the plates. The number of plaquesplated was 500,000. The insert fragment of pCRIIZnf was reamplified and50 ng were end labelled using T4 Polynucleotide Kinase (as described inSambrook et al 1990).

The filters were prehybridised using 0.25% (w/v) Marvel, 5×SSPE and 0.1%(w/v) SDS at 42° C. for 4 hours. The solution in the filters was thenreplaced with fresh solution and the denatured probe added. Thehybridisation was carried out overnight at 42° C. after which thefilters were washed in 6×SSC+0.1% (w/v) SDS at 42° C. followed byanother wash at 55° C. The filters was exposed to X-ray film (Kodak) for48 hours before processing.

The developed film indicated the presence of one strong positive signalwhich was plaque purified and further characterised. The lambda ZAP IIphage was in vivo excised (see Stratagene Manual) and the sequencedetermined of the resulting plasmid DNA. The clone known as pSK19R (or19R) contained a 1.933 kilobase (kb) cDNA fragment with an open readingframe of 467 amino acids (FIG. 2). pSK19R was deposited with the NCIMBon 20 Jun. 1995 and has been accorded the deposit No. NCIMB 40743.

Further analysis of pSK19R revealed that a 340 bp EcoRI fragment mappingat the 5′ end of pSK19R has strong and significant similarities to aDrosophila cDNA encoding glyceraldehyde-3-phosphate dehydrogenase. Inorder to isolate the correct 5′ end sequence belonging to Heliothis, therandom primed library was re-screened using a probe containing the 5′end of the pSK19R belonging to Heliothis ecdysone receptor. The probewas made by PCR using the sense oligonucleotide HecRH3C (5′aattaagcttccaccatgccgttaccaatgccaccgaca 3′; SEQ ID NO:21) and antisenseoligonucleotide HecrNdeI (5′ cttcaaccgacactcctgac 3′; SEQ ID NO: 22).The PCR was carried out as described by Hirst et al., 1992), where theamount of radioisotope used in the labelling was 50 μCi of a ³²P-dCTPand the PCR was cycled for 1 minute at 94° C., 1 minute at 60° C. and 1minute at 72° C. for 19 cycles. The resulting 353 bp radio labelled DNAfragment was denatured and added to prehybridised filters as describedfor the isolation of pSK19R. The library filters were made from 15plates each containing 50,000 pfu. The library filters were hybridisedat 65° C. and washed in 3×SSPE+0.1% SDS at 65° C. twice for 30 minuteseach. The filters were further washed with 1×SSPE+0.1% SDS for 30minutes and exposed to X-ray film (Kodak) overnight. The film wasdeveloped and 16 putative positive plaques were picked. The plaques werere-plated and hybridised under the exact same conditions as the primaryscreen resulting in only one strong positive. The strong positive wasconsistently recognised by the probe and was plaque purified and in vivoexcised. The resulting plasmid pSK16.1 was sequenced SEQ ID NO: 3) whichrevealed that the 5′ end of the clone extended by 205 bp and at the 3′end by 653 bp and resulting in a DNA insert of 2.5 kb. Conceptualtranslation of the 205 bp yielded 73 amino acids with high similarity tothe Drosophila, Aedes aegypti, Manduca and Bombyx sequences of theecdysone receptor B1 isoform. However, the whole of the 5′ end sequenceis not complete since a Methionine start site was not found with a stopcodon in frame 5′ of the methionine. In order to isolate the remainderof the 5′ end coding sequences a 5′ RACE protocol (Rapid Amplificationof cDNA Ends) was carried out using the BRL-GIBCO 5′ RACE Kit. Two typesof cDNA were synthesised where the first one used a specificoligonucleotide:

16PCR2A 5′ cagctccaggccgccgatctcg 3′ (SEQ ID NO: 23)

and the second type used random hexamers (oligonucleotide containing 6random nucleotides). Each cDNA was PCR amplified using theoligonucleotides anchor primer: BRL-GIBCO 5′cuacuacuacuaggccacgcgtcgactagtacgggiigggiigggiig 3′ (SEQ ID NO: 24) and16PCR2A and cycled for 1 minute at 94° C., 1 minute at 60° C. and 1minute at 72° C. for 35 cycles. The reaction conditions were 20 mMTris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 400 nM of each anchor and16PCR2A primers, 200 mM dNTPs (dATP, dCTP, dGTP and dTTP) and 0.02 U/mlTaq DNA polymerase. Dilutions of 1:50 of the first PCR reactions weremade and 1 ml was used in a second PCR with oligonucleotides UAP:(Universal Amplification Primer) 5′ caucaucaucauggccacgcgtcgactagtac 3′(SEQ ID NO: 25)and 16RACE2: (5′ acgtcacctcagacgagctctccattc 3′ (SEQ ID NO: 26).

The conditions and cycling were the same as those followed for the firstPCR. Samples of each PCR were run and a Southern blot carried out whichwas probed with a 5′ specific primer: (16PCR1; 5′cgctggtataacaacggaccattc 3′ (SEQ ID NO. 27).

This primer is specific for the 5′ most sequence of pSK16.1 and washybridised at 55° C. using the standard hybridisation buffer. The filterwas washed at 55° C. 3 times in 3×SSPE+0.1% SDS and exposed to X-rayfilm for up to 6 hours. The developed film revealed bands recognised bythe oligonucleotide migrating at 100 bp and 500 bp (relative to themarkers). A sample of the PCR reaction (4 in total) was cloned into thepCRII vector in the TA cloning kit (Invitrogen). Analysis of 15 clonesfrom 4 independent PCRs yielded sequence upstream of pSK16.1 (FIG. 4).

Translation of the ORF results in a 575 amino acid protein with highsimilarity in the DNA and ligand binding domains when compared to theecdysone receptor sequences of Drosophila, Aedes aegypti, Chironomustentans, Manduca sexta and Bombyx mori (FIG. 5). Interestingly, theN-terminal end of the Heliothis sequence has an in frame methioninestart which is 20 amino acids longer that that reported for Drosophila,Aedes aegypti and Manduca sexta. However, the extended N-terminal end inthe Heliothis EcR does not have similarity to that of Bombyx mori.Finally, the C-terminal ends of the different B1 isoform ecdysonereceptor sequences diverge and do not have significant similarity.

C. Northern Blot Analysis

The sequence identified by screening the library is expected to beexpressed in tissues undergoing developmental changes, thus mRNA fromdifferent developmental stages of H. virescens were was isolated and aNorthern blot produced. The mRNAs were isolated from eggs, 1st, 2nd,3rd, 4th and 5th instar larvae, pupae and adults. The Northern blot washybridised with an NdeI/XhoI DNA fragment from pSK19R encompassing the3′ end of the DNA binding domain through to the end of the ligandbinding domain. The hybridisation was carried out in 1% (w/v) Marvel,5×SSPE, 0.1% (w/v) SDS at 65° C. for 18 to 24 hours. The filters werewashed in 3×SSPE+0.1% (w/v) SDS and 1×SSPE+0.1% (w/v) SDS at 65° C. Thefilter was blotted dry and exposed for one to seven days. The generecognises two transcripts (6.0 and 6.5 kb) which appear to be expressedin all stages examined, however, the levels of expression differ indifferent stages. It should be noted that the same two transcripts arerecognised by probes specific to the DNA binding domain and the ligandbinding domain, indicating that the two transcripts arise from the samegene either by alternative splicing or alternative use ofpolyadenylation sites.

In summary, adult and 5th instar larvae have lower levels of expressionwhile all other tissues have substantial levels of expression.

EXAMPLE II Expression of Heliothis Ecdysone Receptor in Mammalian Cells

To demonstrate that the cDNA encodes a functional ecdysone receptor,effector constructs were generated containing the HEcR under the controlof the CMV (cytomegalovirus) promoter, and the DNA expressed inmammalian cells.

A. Effector Constructs

A first mammalian expression plasmid was constructed by placing aHindIII/NotI pSK19R fragment into the pcDNA3 HindIII/NotI vectorresulting in pcDNA319R (FIG. 7).

A second effector plasmid was constructed wherein the non-coding regionof the cDNA 19R was deleted and a consensus Kozak sequence introduced.The mutagenesis was carried out by PCR amplifying a DNA fragment withthe oligo HecRH3C: 5′aattaagcttccaccatgccgttaccaatgccaccgaca 3′ (SEQ IDNO: 21) containing a unique HindIII restriction enzyme recognition sitefollowed by the mammalian Kozak consensus sequence, and HecRNdeI:5′cttcaaccgacactcctgac 3′ (SEQ ID NO: 22).

The resulting 353 bp PCR fragment was restriction enzyme digested withHindIII and NdeI, gel purified and ligated with 19R NdeI/NotI fragmentinto a pcDNA3 HindIII/NotI vector resulting in pcDNA3HecR.

A third effector construct was made with the 5′ end sequences of pSK16.1by PCR. The PCR approach involved PCR amplifying the 5′ end sequencesusing a 5′ oligonucleotide containing a HindIII restriction cloningsite, the Kozak consensus sequence followed by nucleotide sequenceencoding for a Methionine start and two Arginines to be added to the 5′end of the amplified fragment: (16H3K 5′attaagcttgccgccatgcgccgacgctggtataacaacggaccattc 3′; SEQ ID NO: 28), the3′ oligonucleotide used was HecrNdeI. The resulting fragment wasrestriction enzyme digested, gel purified and subcloned with anNdeI/NotI 19R fragment into pcDNA3 NdeI/NotI vector. The plasmid wasnamed pcDNA3H3 KHEcR.

A fourth effector construct was produced which contains the extendedN-terminal end sequence obtained from the 5′ RACE experiment. Thus, aPCR approach was followed to introduce the new 5′ end sequences inaddition to a consensus Kozak sequence and a HindIII unique cloningsequence. The sense oligonucleotide used was RACEH3K: 5′attaagcttgccgccatgtccctcggcgctcgtggatac 3′ (SEQ ID NO: 29), while theantisense primer was the same as that used before (HecrNdeI). Thecloning strategy was the same as used for the pcDNA3H3KHEcR to give riseto pcDNA3RACEH3KHEcR.

The PCR mutagenesis reactions were carried out in the same manner forall constructs. The PCR conditions used were 1 minute at 94° C., 1minute at 60° C. and 1 minute at 72° C. for 15 cycles. The reactionsconditions were 50 mM Tris-HCl (pH 8.4), 25 mM KCl, 200 mM dNTPs (dATP,dCTP, dGTP and dTTP), 200 nM of each oligonucleotide and 2.5U/Reactionof Taq DNA polymerase. For each construct at least 5 independent PCRreactions were carried out and several clones were sequenced to insurethat at least one is mutation free.

B. Reporter Construct

The reporter plasmid to be co-transfected with the expression vectorcontained 4 copies of the Hsp27 ecdysone response element (Riddihoughand Pelham, 1987) fused to β-globin promoter and the β-Galactosidasegene. The tandem repeats of the ecdysone response element weresynthesised as two complementary oligonucleotides which when annealedproduced a double stranded DNA molecule flanked by an SpeI site at the5′ end and a ClaI site at the 3′ end:

Recr3A: (SEQ ID NO: 30) 5′ctagtagacaagggttcaatgcacttgtccaataagcttagacaagggttcaatgcacttgtccaatgaattcagacaagggttcaatgcacttgtccaatctgcagagacaagggttcaatgcacttgtccaatat 3′ Recr3B: (SEQID NO: 31) 5′cgatattggacaagtgcattgaacccttgtctctgcagattggacaagtgcattgaacccttgtctgaattcattggacaagtgcattgaacccttgtctaagcttattggacaagtgcattgaacccttgtcta 3′.

The resulting 135 bp DNA fragment was ligated to the vector pSWBGALSpeI/ClaI resulting in pSWREcR4 (FIG. 8). The co-transfection of the twoplasmids should result in B-galactosidase activity in the presence ofligand. The experiment relies upon the presence of RXR (a homologue ofultraspiracle) in mammalian cells for the formation of an activeecdysone receptor.

C. Mammalian Transfection Methods

Transfections of mammalian cell lines (CHO-K1 Chinese hamster ovary)ATCC number CCL61 or cos-1 (Monkey cell line) were performed usingeither calcium phosphate precipitation (Gorman, Chapter 6 of “DNAcloning: a practical approach. Vol 2 D. M. Glover ed/.(1985) IRL Press,Oxford) or using LipofectAMINE (Gibco BRL Cat. No. 18324–012, followingmanufacturer's instructions). Human Epithelial Kidney 293 cells weretransfected using analogous methods.

D. Results—Native HEcR Drives Transient Reporter Gene Expression inMammalian Cells

Co-transfection of pcDNA3H3 KHEcR (Effector) and reporter constructsinto Human Epithelial Kidney 293 cells (HEK293) in the presence ofeither Muristerone A or RH5992 resulted in a 2–3 fold induction ofreporter activity compared to the no chemical controls (FIG. 9). TheHEK293 cells were used since they are known to have constitutive levelsof αRXR which have been demonstrated to be necessary for Drosophila EcRactivation by Muristerone A (Yao et al., 1993). Moreover, to furtherinvestigate the need for RXR interactions, a αRXR was co-transfectedinto HEK293 cells (along with the effector and reporter) resulting in a9-fold induction of reporter activity compared to the untreated cells(FIG. 9). The co-transfection of αRXR with reporter and effectorincreased by four fold the reporter activity compared to cellstransfected with effector and reporter alone. Induction was observedboth in the presence of either Muristerone A or RH5992. These dataclearly demonstrate that the cDNA HEcR encodes a functional ecdysonereceptor. Moreover, the ability of HEcR to complex with αRXR and bindMuristerone A or RH5992 provide evidence for the usage of the entireHEcR as a component of a mammalian gene switch. In particular, it offersthe advantage of reducing uninduced expression of target gene sinceecdysone receptor and response elements are not present in mammaliancells.

EXAMPLE III Chimeric Constructs and Ligand Validation in MaizeProtoplasts

In order to apply the ecdysone receptor as an inducible system it wasdeemed necessary to simplify the requirements of the system by avoidingthe need of a heterodimer formation to obtain an active complex. Theglucocorticoid receptor is known to form homodimers and chimericconstructs of the glucocorticoid receptor transactivating and DNAbinding domains fused to the ecdysone receptor hinge and ligand bindingdomains have been shown to be active as homodimers in mammalian cells inthe presence of Muristerone A (an ecdysone agonist) (Christopherson etal., 1992). However, the chimeric receptor is not responsive to20-hydroxyecdysone (Christopherson et al., 1992).

The analysis of the activation of the glucocorticoid/Heliothis ecdysonechimeric receptor entailed the production of two other control effectorconstructs. The first one of the constructs contained the intactglucocorticoid receptor while the second one contained aglucocorticoid/Drosophila ecdysone chimeric receptor.

A. Effector Constructs

(i) Glucocorticoid Receptor Maize Expression Construct

The glucocorticoid receptor DNA for the Maize transient expressionconstruct was produced via the polymerase chain reaction (PCR) of HumanFibrosarcoma cDNA (HT1080 cell line, ATCC#CC1121) library (Clontech seeHollenberg et al., 1985). The PCR approach taken was to amplify the 2.7kb fragment encoding the glucocorticoid receptor in two segments. Thefirst segment entails the N-terminal end up to and including the DNAbinding domain while the second fragment begins with the hinge region(amino acid 500) thought to the end of the reading frame. Thus, the PCRprimer for the N-terminal end segment was designed to contain an EcoRIsite and the Kozak consensus sequence for translation initiation:GREcoRI 5′ attgaattccaccatggactccaaagaatcattaactc 3′ (SEQ ID NO: 32).

The 3′ end primer contains an XhoI site in frame with the reading frameat amino acid 500 of the published sequence: GRXhoI 5′gagactcctgtagtggcctcgagcattccttttatttttc 3′ (SEQ ID NO: 33).

The second fragment of the glucocorticoid receptor was produced with a5′ end oligonucleotide containing an XhoI site in frame with the openreading frame at the beginning of the hinge region (amino acid 500):GRHinge 5′ attctcgagattcagcaggccactacaggag 3′ (SEQ ID NO: 34) while the3′ end oligonucleotide contained an EcoRI site 400 bp after the stopcodon: GRStop; 5′ attgaattcaatgctatcgtaactatacaggg 3′ (SEQ ID NO: 35).

The glucocorticoid receptor PCR was carried out using Vent polymerase(Biolabs) under hot start conditions followed by 15 cycles of denaturing(94° C. for 1 minute), annealing (66° C. for 1 minute) and DNA synthesis(72° C. for 3 minutes). The template was produced by making first strandcDNA as described in the TA cloning kit (Invitrogen) after which the PCRwas carried out in 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM TRIS-HCl pH 8.8, 2mM MgSO₄, 0.1% (v/v) Triton X-100, 200 mM dNTPs, 100 ng of each Primerand 2 U of Vent Polymerase. The PCR products were restriction enzymedigested with EcoRI and XhoI and subcloned into pBluescript SK (pSK)EcoRI. The resulting plasmid pSKHGI was sequenced and found to lack anymutations from the published sequences (apart from those introduced inthe PCR primers) (Hollenberg et al., 1985).

The 2.7 kb EcoRI fragment was subcloned into the vector pMF6PAT EcoRIresulting in pMF6HGIPAT (FIG. 10).

(ii) Maize Expression Construct Containing a Glucocorticoid/DrosophilaEcdysone Chimeric Receptor.

The glucocorticoid receptor portion of the chimeric receptor wasisolated from pSKHGI by producing a 1.5 kb BamHI/XhoI restrictionfragment containing the N-terminal end up to and including the DNAbinding domain.

The Drosophila ecdysone receptor portion was isolated through PCR offirst stand cDNA prepared from Drosophila adult mRNA. The PCR wascarried out using a 5′ oligonucleotide containing a SalI site (i.e.Drosophila ecdysone receptor contains a XhoI site at the end of theligand binding domain) which starts at the beginning of the hingeregion: amino acid 330:

Ecr8 attgtcgacaacggccggaatggctcgtcccggag 3′ (SEQ ID NO: 36).

The 3′ end oligonucleotide contains a BamHI site adjacent to the stopcodon:

EcRstop 5′ tcgggctttgttaggatcctaagccgtggtcgaatgctccgacttaac 3′ (SEQ IDNO: 37).

The PCR was carried out under the conditions described for theamplification of the Glucocorticoid receptor and yielded a 1.6 kbfragment. The fragment was introduced into pSK SalI/BamHI and thesequence determined and compared to the published one (Koelle et al.,1991).

The maize transient expression plasmid was produced by introducing intopMF6 BamHI vector the 1.5 kb BamHI/XhoI glucocorticoid receptor fragmentand the 1.6 kb SalI/BamHI Drosophila receptor portion to yield thechimeric plasmid pMF6GREcRS (FIG. 9).

(iii) Construction of the Glucocorticoid/Heliothis Ecdysone ChimericReceptor Maize Transient Expression Plasmid.

The Glucocorticoid receptor portion of the chimera was produced asdescribed in Example II(ii). The production of the Heliothis ecdysonereceptor portion involves the introduction of a SalI recognition site atthe DNA binding/hinge domain junction (amino acid 229). The addition ofthe SalI site:

Hecrsal 5′attgtcgacaaaggcccgagtgcgtggtgccggag 3′ (SEQ ID NO: 38)

was achieved via PCR mutagenesis making use of a unique AccI site 107 bpdownstream of the junction point (or 1007 bp relative to SEQ ID NO: 4):Hecracc 5′ tcacattgcatgatgggaggcatg 3′ (SEQ ID NO: 39).

The PCR was carried out using Taq polymerase (2.5 U) in a reactionbuffer containing 100 ng of template DNA (pSK19R), 100 ng of Hecrsal andHecracc, 20 mM TRIS-HCl pH 8.4, 50 mM KCl, 1 mM MgCl₂, 200 mM dNTPs. Thereaction was carried out with an initial denaturation of 3 minutesfollowed by 15 cycles of denaturation (1 minute at 94° C.), annealing (1minute at 60° C.) and DNA synthesis (1 minute at 72° C.). The DNA wasrestriction enzyme digested and subcloned into pSK SalI/SacI with the1.2 kb AccI/SacI 3′ end HecR fragment to yield pSK HeCRDEF (orcontaining the hinge and ligand binding domains of the Heliothisecdysone receptor). The construction of the maize transient expressionplasmid containing the Glucocorticoid/Heliothis ecdysone chimericreceptor involved the ligation of pMF6 EcoRI/SacI with the 1.5 kbEcoRI/XhoI fragment of Glucocorticoid receptor N-terminal end and the1.2 kb SalI/SacI fragment of pSk HEcRDEF to yield pMF6GRHEcR (FIG. 10).

B. Reporter Plasmids

Two reporter plasmids were made by inserting the into p221.9 or p221.10BamHI/HindIII vectors two pairs or oligonucleotides containing sixcopies of the glucocorticoid response element (GRE). The two sets ofoligonucleotides were designed with restriction enzyme recognition sitesso as to ensure insertion of the two pairs in the right orientation. Thefirst oligonucleotide pair GRE1A/B is 82 nucleotides long and whenannealed result in a DNA fragment flanked with a HindIII site at the 5′end and a SalI site at the 3′ end:

GRE1A (SEQ ID NO: 40) 5′agcttcgactgtacaggatgttctagctactcgagtagctagaacatcctgtacagtcgagtagctagaacatcctgtacag3′ GRE1B (SEQ ID NO: 41) 5′tcgactgtacaggatgttctagctactcgactgtacaggatgttctagctactcgagtcgctagaacatcctgtacagtcga3′.

The second pair of oligonucleotides is flanked by a SalI site at the 5′end and a BamHI site at the 3′ end:

GRE2A (SEQ ID NO: 42) 5′tcgactagctagaacatcctgtacagtcgagtagctagaacatcctgtacagtcgagtagctagaacatcctgtacag3′ GRE2B (SEQ ID NO: 43) 5′gatcctgtacaggatgttctagctactcgactgtacaggatgttctagctactcgactgtacaggatgttctagctag3′.

The resulting plasmids were named p221.9GRE6 (FIG. 13) and p221.10GRE6(FIG. 14) (used in later Example). The difference between p221.9 andp221.10 plasmids is that p221.9 contains the −60 35SCaMV minimalpromoter while p221.10 (p221.10GRE6) contains the −46 35SCaMV minimalpromoter.

C. Method

Protoplasts were isolated from a maize suspension culture derived fromBE70×A188 embryogenic callus material, which was maintained bysubculturing twice weekly in MS0.5_(mod). (MS medium supplemented with3% sucrose, 690 mg/l proline, 1 g/l myo-inositol, 0.2 g/l casein acidhydrolysate, 0.5 mg/l 2,4-D, pH 5.6). Cells from suspensions two dayspost subculture were digested in enzyme mixture (2.0% Cellulase RS, 0.2%Pectolyase Y23, 0.5 M Mannitol, 5 mM CaCl₂.2H₂O, 0.5% MES, pH 5.6, ˜660mmol/kg) using ˜10 ml/g cells, incubating at 25° C., dim light, rotatinggently for ˜2 hours. The digestion mixture was sieved sequentiallythrough 250 μm and 38 μm sieves, and the filtrate centrifuged at 700 rpmfor 3.5 minutes, discarding the supernatant. The protoplasts wereresuspended in wash buffer (0.358 M KCl, 1.0 mM NH₄NO₃, 5.0 mMCaCl₂.2H₂O, 0.5 mM KH₂PO₄, pH 4.8, ˜670 mmol/kg) and pelleted as before.This washing step was repeated. The pellet was resuspended in washbuffer and the protoplasts were counted. Transformation was achievedusing a Polyethylene glycol method based on Negrutiu et al. Protoplastswere resuspended at 2×10⁶/ml in MaMg medium (0.4 M Mannitol, 15 mMMgCl₂, 0.1% MES, pH 5.6, ˜450 mmol/kg) aliquotting 0.5 ml/treatment(i.e. 1×10⁶ protoplasts/treatment). Samples were heat shocked at 45° C.for 5 minutes then cooled to room temperature. 10 μg each of p221.9GRE6and pMF6HR1PAT (GR) (1 mg/ml)/treatment were added and mixed in gently,followed by immediate addition of 0.5 ml warm (˜45° C.) PEG solution(40% PEG 3,350 MW in 0.4 M Mannitol, 0.1 M Ca(NO₃)₂, pH 8.0), which wasmixed in thoroughly but gently. Treatments were incubated at roomtemperature for 20–25 minutes, then 5 ml 0.292 M KCl (pH 5.6, ˜530mmol/kg) was added step-wise, 1 ml at a time, with mixing. Thetreatments were incubated for a further 10–15 minutes prior to pelletingthe protoplasts by centrifuging as before. Each protoplast treatment wasresuspended in 1.5 ml culture medium (MS medium, 2% sucrose, 2 mg/l2,4-D, 9% Mannitol, pH 5.6, ˜700 mmol/kg)+/−0.0001 M dexamethasone(glucocorticoid). The samples were incubated in 3 cm dishes at 25° C.,dark, for 24–48 hours prior to harvesting. Fluorometric assays for GUSactivity were performed with the substrate4-methylumbelliferyl-D-glucuronide using a Perkin-Elmer LS-35fluorometer (Jefferson et al., 1987). Protein concentrations of tissuehomogenates were determined by the Bio-Rad protein assay (Bradford,1976). The method was repeated for each effector construct.

D. Results

1. Reporter Gene Assay

A reporter gene construct (p221.9GRE6) was generated containing the GUSreporter gene under the control of a −60 CaMV 35S promoter with 6 copiesof the glucocorticoid response element. To test this construct wasfunctional in maize protoplasts a co-transformation assay was performedwith the reporter construct p221.9GRE6 and the effector constructpMF6HR1PAT (GR) construct containing the entire glucocorticoid receptor.

FIG. 15 shows that Reporter p221.9GRE6 alone or reporter plus effectorpMF6HR1PAT (GR) with no activating chemical gave no significantexpression. When reporter plus effector were co-transformed into maizeprotoplasts in the presence of 0.0001 M dexamethasone (glucocorticoid),a significant elevation of marker gene activity was observed (FIG. 15).The response is specific to glucocorticoid as the steroid Muristerone Adoes not lead to induced levels of expression. These studies clearlyshow the reporter gene construct p221.9GRE6 is capable of monitoringeffector/ligand mediated gene expression.

2. Chimeric Ecdysone Effector Constructs Mediate Inducible Expression inMaize Transient Protoplasts Assays

A chimeric effector plasmid pMF6GREcRS was constructed, containing theligand binding domain from the Drosophila ecdysone receptor and the DNAbinding and transactivation domain from the glucocorticoid receptor. Toconfirm the reporter gene construct p221.9GRE6 could respond to achimeric ecdysone effector construct, a series of co-transformation intomaize protoplasts was performed.

FIG. 16 shows that reporter (p221.9GRE6) alone or reporter plus effector(pMF6GREcRS) with no activating chemical, gave no significant expressionin maize protoplasts. When reporter plus effector were co-transformedinto maize protoplasts in the presence of 100 μM Muristerone A, asignificant elevation of marker gene activity was observed. The responsewas specific to Muristerone A, as the steroid dexamethasone did not leadto induced levels of expression. These studies clearly showed thereporter gene construct p221.9GRE6 is capable of monitoring chimericecdysone effector/ligand mediated gene expression.

A second chimeric effector construct pMF6GRHEcR, was generatedcontaining the ligand binding domain from Heliothis ecdysone receptor.When co-transformed into maize protoplasts with the reporter plasmidp221.9GRE6, no response to 100 μM Muristerone or 100 μM dexamethasonewas observed (FIG. 17). These data clearly show the Drosophila andHeliothis ligand binding domains exhibit different properties.

When the effector plasmid pMF6GREcRS, containing the ligand bindingdomain from Drosophila, was tested with the reporter p221.9GRE6 inpresence of the non-steroidal ecdysone agonists RH5849 and RH5992(mimic), no chemical induced reporter gene activity was observed (FIGS.18 and 19).

When the effector plasmid pMF6GRHEcR, containing the ligand bindingdomain from Heliothis, was tested with the reporter p221.9GRE6 inpresence of the non-steroidal ecdysone agonists RH5992 (mimic),significant chemical induced reporter gene activity was observed (FIG.20). These data demonstrate the ligand binding domain from Heliothis hasdifferent properties to the Drosophila receptor in that the formerresponded to the non-steroidal ecdysteroid agonist RH5992. FIG. 21demonstrates the effector plasmid pMF6GRHEcR confers RH5992 dependantinducibility on the reporter p221.9GRE6 in a dose responsive manner.Induction was observed in a range from 1 μM–100 μM RH5992.

EXAMPLE IV Testing of Effector Vectors in Tobacco Protoplasts

The experiments carried out in the previous example demonstrated thespecific effect of RH5992 (mimic) on pMF6GRHEcR in maize protoplasts. Itis the aim in this example to show the generic application to plants ofthe glucocorticoid/Heliothis ecdysone chimeric receptor switch system.Tobacco shoot cultures cv. Samsun, were maintained on solidified MSmedium +3% sucrose in a controlled environment room (16 hour day/8 hournight at 25° C., 55% R.H), were used as the source material forprotoplasts. Leaves were sliced parallel to the mid-rib, discarding anylarge veins and the slices were placed in CPW13M 13% Mannitol, pH 5.6,˜860 mmol/kg) for ˜1 hour to pre-plasmolyse the cells. This solution wasreplaced with enzyme mixture (0.2% Cellulase R10, 0.05% Macerozyme R10in CPW9M (CPW13M but 9% Mannitol), pH 5.6, ˜600 mmol/kg) and incubatedin the dark at 25° C. overnight (˜16 hours). Following digestion, thetissue was teased apart with forceps and any large undigested pieceswere discarded. The enzyme mixture was passed through a 75 μm sieve andthe filtrate was centrifuged at 600 rpm for 3.5 minutes, discarding thesupernatant. The pellet was resuspended in 0.6 M sucrose solution andcentrifuged at 600 rpm for 10 minutes. The floating layer of protoplastswas removed using a Pasteur pipette and diluted with CPW9M (pH 5.6, ˜560mmol/kg). The protoplasts were again pelleted by centrifuging at 600 rpmfor 3.5 minutes, resuspended in CPW9M and counted. A modified version ofthe PEG-mediated transformation above was carried out. Protoplasts wereresuspended at 2×10⁶/ml in MaMg medium and aliquotted using 200μl/treatment (i.e. 4×10⁵ protoplasts/treatment). 20 μg each ofpMF6GRHEcRS and p221.9GRE6 DNA (1 mg/ml) were added followed by 200 μlPEG solution and the solutions gently mixed. The protoplasts were leftto incubate at room temperature for 10 minutes before addition of 5 mlMSP19M medium (MS medium, 3% sucrose, 9% Mannitol, 2 mg/l NAA, 0.5 mg/lBAP, pH 5.6, ˜700 mmol/kg)+/−10 μM RH5992. Following gentle mixing, theprotoplasts were cultured in their tubes, lying horizontally at 25° C.,light. The protoplasts were harvested for the GUS assay after ˜24 hours.

A. Effector Construct

(i) Construction of a Dicotyledonous Expression Vector

The vector produced is a derivative of pMF6. pMF6GREcRS was restrictionenzyme digested with PstI to produce 3 fragments namely, 3.4 (AdhIntronless pMF6), 3.2 (GREcRS) and 0.5 (Adh intron I) kb. Isolation andreligation of the 3.4 and 3.2 kb fragments resulted in pMF7GREcRS (FIG.22). pMF7GREcRS was restriction enzyme digested with EcoRI/SacIresulting in the 3.4 kb pMF7 EcoRI/SacI vector which when isolated andpurified was ligated to a 1.5 kb EcoRI/XhoI N-terminal end of theglucocorticoid receptor and the 1.2 kb SalI/SacI Heliothis ecdysoneC-terminal end sequences to produce pMF7GRHEcR (FIG. 23).

B. Reporter Plasmid

The reporter plasmids constructed for the maize transient experimentswere the same as those used without alteration in the tobacco leafprotoplast transient expression experiments.

C. Results—Chimeric Ecdysone Effector Constructs Mediate InducibleExpression in Tobacco Transient Protoplast Assays

Experiments were performed to demonstrate that the effector plasmidpMF6GRHEcR can confer chemical dependant inducible expression on thereporter p221.9GRE6 in tobacco mesophyll protoplasts.

FIG. 24 shows that reporter (p221.9GRE6) alone or reporter plus effector(pMF7GRHEcR) with no activating chemical, gave no significant expressionin tobacco protoplasts. When reporter plus effector were co-transformedinto tobacco protoplasts in the presence of 10 μM RH5992, a significantelevation of marker gene activity was observed. These data show achimeric ecdysone effector construct, containing the Heliothis ligandbinding domain can confer non-steroidal ecdysteroid dependant expressionon reporter gene constructs in both monocotyledonous and dicotyledonousspecies.

EXAMPLE V Chimeric Activity in Mammalian Cells

A. Effector Constructs

(i) Construction of Glucocorticoid/Heliothis Ecdysone Chimeric Receptor.

The mammalian expression vector used in this experiment was pcDNA3(Invitrogen). The GRHEcR 2.7 kb BamHI DNA fragment (isolated frompMF6GRHEcR) was introduced into the pcDNA3 BamHI vector. Therecombinants were oriented by restriction enzyme mapping. The DNAsequence of the junctions was determined to ensure correct orientationand insertion (pcDNA3GRHEcR, FIG. 25).

B. Reporter Construct

The reporter plasmid for mammalian cell system was produced by takingpSWBGAL plasmid and replacing the CRESW SpeI/ClaI fragment for asynthetic 105 bp DNA fragment containing 4 copies of the glucocorticoidresponse element (GRE) and flanked by SpeI at the 5′ end and AflII atthe 3′ end.

The oligonucleotides were synthesised using the sequences:

GREspeI (SEQ ID NO: 44)5′ctagttgtacaggatgttctagctactcgagtagctagaacatcctgtacagtcgagtagctagaacatcctgtacagtcgagtagctagaacatcctgtacac 3′; GREafl2 (SEQ ID NO: 45)5′ttaagtgtacaggatgttctagctactcgactgtacaggatgttctagctactcgactgtacaggatgttctagctactcgagtagctagaacatcctgtacaa 3′.

The two oligonucleotides were purified annealed and ligated to pSWBGALSpeI/AflII to produce pSWGRE4 (FIG. 26).

C. Results—Chimeric HEcR Drives Transient Reporter Gene Expression inMammalian Cells

No expression was detected when a reporter gene construct pSWGRE4,comprising of a minimal β-globin promoter containing four copies of theglucocorticoid response element, fused to a β-galactosidase reportergene, was introduced into CHO cells. Similarly, no expression wasdetected when pSWGRE4 and an effector plasmid pcDNA3GRHEcR, containingthe transactivation and DNA binding domain from the glucocorticoidreceptor and the ligand binding domain from the Heliothis ecdysonereceptor, under the control of the CMV promoter were co-transformed intoCHO-K1 or HEK293 cells. When co-transformed CHO (FIG. 27) and HEK293cells (FIG. 28) were incubated in the presence of the non-steroidalecdysone agonists RH5992 (mimic), significant chemical induced reportergene activity was observed. Equally, induction of reporter activity wasobserved when HEK293 cells transfected with pcDNA3GRHEcR and reporterwere treated with Muristerone A (FIG. 28).

EXAMPLE VI Screening System Allows New Chemical Activators and ModifiedLigand Binding Domains to be Tested in Mammalian Cells

The basis of a screening system is in place after the demonstration thatthe chimeric receptor was activated in the presence of RH5992. A screenwas carried out using CHO cells transiently transfected with bothpSWGRE4 (reporter) and pcDNA3GRHEcR (effector) constructs. In the firstinstance 20 derivatives compounds of RH5992 were screened. It wasobserved that 7 out of the 20 compounds gave an increased reporter geneactivity compared to untreated cells. A second screen was carried out inwhich 1000 randomly selected compounds were applied to transientlytransfected CHO cells. Two compounds were found to activate reportergene activity above that from the untreated controls. The second screensuggest that this cell based assay is a robust and rapid way to screen asmall library of compounds, where a thousand compounds can be putthrough per week.

EXAMPLE VII Stably Transformed Tobacco Plants

A. Stable Tobacco Vectors

The components of the stable Tobacco vectors were put together inpBluescript prior to transfer into the binary vector. The production ofstable transformed plants entails the production of a vector in whichboth components of the switch system (i.e. effector and reporter) areplaced in the same construct to then introduce into plants.

The methodology described below was used to produce four differentstable Tobacco vectors. The method involves three steps:

-   1. pbluescript SK HindIII/EcoRI vector was ligated to either    GRE6-4635SCaMVGUSNOS HindIII/EcoRI (from p221.10GRE6) or    GRE6-6035SCaMVGUSNOS HindIII/EcoRI (from p221.9GRE6) resulting in    plasmid pSK-46 and pSK-60.-   2. This step involves the addition of the chimeric receptor    (35SGRHEcRNOS or 35SGRVP16HEcRNOS) to pSK-60 or pSK-46. Thus a    pSK-60 (or pSK-46) XbaI vector was ligated with either the 3.4 kb    35SGRHEcRNOS XbaI or the 3.0 kb 35SGRVP16HEcRNOS XbaI DNA fragment    to produce pSKES1 (pSKGRE6-6035SCaMVGUSNOS-35SGRHEcRNOS), pSKES2    (pSKGRE6-4635 SCaMVGUSNOS-35 SGRHEcRNOS), pSKES3    (pSKGRE6-6035SCaMVGUSNOS-35SGRVP16HEcRNOS) and pSKES4    (pSKGRE6-4635SCaMVGUSNOS-35SGRVP16HEcRNOS).-   3. Transfer from pBluescript based vectors to binary vectors. The    transfer of the ES1 (FIG. 29) ES2 (FIG. 30), ES3 (FIG. 31) or ES4    (FIG. 32) DNA fragments into the binary vector JR1 involves five    steps:    -   (i) Restriction enzyme digestion of pSKES1 (ES2, ES3, and ES4)        with ApaI and NotI to liberate the insert from the vector        pBluescript.    -   (ii) The two DNA fragments were BamHI methylated for 2 hours at        37° C. in TRIS-HCl, MgCl, 80 μM SAM (S-adenosylmethionine) and        20 U of BamHI methylase.    -   (iii) Ligate an ApaI/NotI linker onto the fragment. The linker        was designed to have an internal BamHI site:        -   ApaBNot1 5′ cattggatccttagc 3′ (SEQ ID NO: 46) and        -   ApaBNot2 5′ ggccgctaaggatccaatgggcc 3′ (SEQ ID NO: 47).    -   (iv) Restriction enzyme digest the protected and linkered        fragment with BamHI and fractionate the products on a 1% (w/v)        agarose gel. The protected DNA fragment (5.5 kb) was cut out of        the gel and purified.    -   (v) A ligation of JRI BamHI vector with the protected band was        carried out to produce JRIESI (JRIES2, JRIES3 or JRIES4). The        DNA of the recombinant was characterised by restriction mapping        and the sequence of the junctions determined.

The plant transformation construct pES1, containing a chimeric ecdysonereceptor and a reporter gene cassette, was transferred intoAgrobacterium tumefaciens LBA4404 using the freeze/thaw method describedby Holsters et al. (1978). Tobacco (Nicotiana tabacum cv Samsun)transformants were produced by the leaf disc method (Bevan, 1984).Shoots were regenerated on medium containing 100 mg/l kanamycin. Afterrooting, plantlets were transferred to the glasshouse and grown under 16hour light/8 hour dark conditions.

B. Results—Chimeric Ecdysone Effector Constructs Mediate InducibleExpression in Stably Tobacco Plants

Transgenic tobacco plants were treated in cell culture by adding 100 μMRH5992 to MS media. In addition seedlings were grown hydroponically inthe presence or absence of RH5992. In further experiments 5 mM RH5992was applied in a foliar application to 8 week old glasshouse growntobacco plants. In the three methods described uninduced levels of GUSactivity were comparable to a wild type control, while RH5992 levelswere significantly elevated.

Ecdysone Switch Modulation and Optimisation

EXAMPLE VIII Yeast Indicator Strains for Primary Screen of ChemicalLibraries

A set of yeast indicator strains was produced to use as a primary screento find chemicals which may be used in the gene switch. The propertiesof the desired chemicals should include high affinity resulting in highactivation but with different physico-chemical characteristics so as toincrease the scope of application of the technology. Moreover, theproduction of this strain also demonstrates the generic features of thisswitch system.

A. Effector Vector

A base vector for yeast YCp15Gal-TEV-112 was generated containing:

-   Backbone—a modified version of pRS315 (Sikorski and Hieter (1989)    Genetics 122, 19–27) a shuttle vector with the LEU2 selectable    marker for use in yeast;-   ADH1 promoter (BamHI-Hind III fragment) and ADH1 terminator (Not    I-Bam HI fragment) from pADNS (Colicelli et al PNAS 86, 3599–3603);-   DNA binding domain of GAL4 (amino acids 1–147; GAL4 sequence is    Laughon and Gesteland 91984) Mol. Cell Biol. 4, 260–267) from pSG424    (Sadowski and Ptashne (1989) Nuc. Acids Res. 17, 7539);-   Activation domain—an acidic activation region corresponding to amino    acids 1–107 of activation region B112 obtained from plasmid pB112    (Ruden et al (1991) Nature 350, 250–252).

The plasmid contains unique Eco RI, Nco I and Xba I sites between theDNA binding domain and activation domains.

Into this vector a PCR DNA fragment of the Heliothis ecdysone receptorcontaining the hinge, ligand binding domains and the C-terminal end wasinserted. The 5′ oligonucleotide is flanked by an NcoI restrictionrecognition site and begins at amino acid 259: HecrNcoI 5′aattccatggtacgacgacagtagacgatcac 3′ (SEQ ID NO: 48).

The 3′ oligonucleotide is flanked by an XbaI site and encodes for up toamino acid 571: HecRXbaI 5′ ctgaggtctagagacggtggcgggcggcc 3′ (SEQ II)NO: 49).

The PCR was carried out using vent polymerase with the conditionsdescribed in Example IA. The fragment was restriction enzyme digestedwith NcoI and XbaI purified and ligated into YCp15GALTEV112 NcoI/XbaIvector to produce YGALHeCRB112 or TEV-B112 (FIG. 34). In order to reduceconstitutive activity of the YGALHeCRB112 plasmid a YGALHeCR plasmid wasproduced in which the B112 activator was deleted by restriction enzymedigesting YGALHeCRB112 with XbaI/SpeI followed by ligation of theresulting vector (i.e. SpeI and XbaI sites when digested producecompatible ends) (TEV-8, FIG. 33). An effector plasmid was constructedwhereby the B112 transactivating domain was excised from YGalHecRB112with XbaI and replaced with the VP16 transactivation domain DNA fragment(encoding amino acids 411 and 490 including the stop codon). Theresulting vector was named YGalHecRVP16 or TEVVP16-3 (FIG. 35).

B. Reporter Construction for Yeast

The S. cerevisiae strain GGY1::171 (Gill and Ptashne (1987) Cell 51,121–126), YT6::171 (Himmelfarb et al (1990) Cell 63, 1299–1309) bothcontain reporter plasmids consisting of the GAL4-responsive GAL1promoter driving the E. coli β-galactosidase gene. These plasmids areintegrated at the URA3 locus. The reporter strain YT6::185 contains thereporter plasmid pJP185 (two synthetic GAL4 sites driving theB-galactosidase gene) integrated at the URA3 locus of YT6 (Himmelfarb etal). (Note—the parental strains YT6 and GGY1 have mutations in the GAL4and GAL80 genes, so the reporter genes are inactive in the absence ofany plasmids expressing GAL4 fusions).

C. Yeast Assay

Standard transformation protocols (Lithium acetate procedure) andselection of colonies by growth of cells on selective media (leucineminus medium in the case of the YCp15Gal-TEV-112 plasmid)—as describedin Guthrie and Fink 1991) Guide to Yeast Genetics and Molecular Biology.Methods in Enzymology Vol. 194 Academic Press and the reporter geneassay is a modification of that described in Ausubel et al., (1993)Current Protocols in Molecular Biology (Wiley) Chapter 13.

D. Results—Automated Screening System Allows New Chemical Activators andModified Ligand Binding Domains to be Tested in Yeast

An effector vector pYGALHEcRB112 has been generated containing a GAL4DNA binding domain, a B112 activation domain and the ligand bindingregion from Heliothis virescens. In combination with a GAL reportervector, pYGALHEcRB112 form the basis of a rapid, high throughput assaywhich is cheap to run. This cell-based assay in yeast (Saccharomycescerevisiae) will be used to screen for novel non-steroidal ecdysoneagonists which may of commercial interest as novel insecticides orpotent activators of the ecdysone gene switch system. The demonstrationof an efficient system to control gene expression in a chemicaldependant manner, forms the basis of an inducible system for peptideproduction in yeast.

The yeast screening system forms the basis of a screen for enhancedligand binding using the lac Z reporter gene vector to quantitativelyassay the contribution of mutation in the ligand binding domain.Alternatively, enhanced ligand binding capabilities or with a selectioncassette where the lac Z reporter is replaced with a selectable markersuch as uracil (URA 3), tryptophan (Trp1) or leucine (Leu2), andhistidine (His). Constructs based on pYGALHEcRB112 with alterations inthe ligand binding domain are grown under selection conditions whichimpair growth of yeast containing the wild type ligand binding domain.Those surviving in the presence of inducer are retested and thensequenced to identify the mutation conferring resistance.

EXAMPLE IX Optimisation of Chimeric Receptor Using a StrongTransactivator

A. Construction of Mammalian Expression Plasmid with Chimeric ReceptorContaining Herpes Simplex VP16 Protein Sequences.

The construction of this chimeric receptor is based on replacing thesequences encoding for the glucocorticoid receptor transactivatingdomain with those belonging to the VP16 protein of Herpes simplex. ThusPCR was used to generate three fragments all to be assembled to producethe chimeric receptor. The PCRs were carried out as described in ExampleII, iii. The first fragment includes the Kozak sequences and methioninestart site of the glucocorticoid receptor to amino acid 152 of theglucocorticoid receptor. The oligonucleotides used for the generation ofthis fragment included an EcoRI site at the 5′ end: GR1A 5′atatgaattccaccatggactccaaagaatc 3′ (SEQ ID NO: 50), and at the 3′ end aNheI restriction enzyme recognition site: GR1B 5′atatgctagctgtgggggcagcagacacagcagtgg 3′ (SEQ ID NO: 51).

The second fragment also belongs to the glucocorticoid receptor andbegins with a NheI site in frame with amino acid 406: GR2A 5′atatgctagctccagctcctcaacagcaacaac 3′ (SEQ ID NO: 52), and ends with aXhoI site at amino acid 500: GR2B 5′ atatctcgagcaattccttttatttttttc 3′(SEQ ID NO: 53).

The two fragments were introduced into pSKEcoRI/SacI in a ligationcontaining GR1A/B EcoRI/NheI, GR2A/B NheI/XhoI and HEcR SalI/SacI (frompSKHEcRDEF) to yield pSKGRDHEcR. The GR sequences and junctions of theligation were found to be mutation free.

The third fragment to be amplified was a sequence between amino acid 411to 490 of the herpes simplex VP16 protein. The amplified fragment wasflanked with SpeI recognition sites. SpeI produces compatible ends tothose of NheI sites. The oligonucleotides used: VP16C 5′attactagttctgcggcccccccgaccgat 3′ (SEQ ID NO: 54) and VP16E 5′aattactagtcccaccgtactcgtcaattcc 3′ (SEQ ID NO: 55) produced a 180 bpfragment which was restriction enzyme digested with SpeI and introducedinto pSKGRΔHEcR NheI vector to produce pSKGRVP16HEcR. The DNA from thelatter was sequenced and found to be mutation free, the junctions werealso shown to be in frame with those of the glucocorticoid receptor.

The 2.2 kb EcoRV/NotI GRVP16HEcR fragment was introduced into a pcDNA3EcoRV/NotI vector resulting in pcDNA3GRVP16HEcR (FIG. 36).

B. Construction of Plant Transient Expression Effector PlasmidsContaining the Chimeric Receptor with VP16 Sequences

The same procedure was carried out to clone the GRVP16HeCR DNA fragmentinto tobacco (pMF7b) and maize (pMF6) expression vectors. A 2.2 kb BamHIDNA fragment was isolated from pcDNA3GRVP16HeCR and ligated in to thepMF6 BamHI (or pMF7b BamHI) vector to produce pMF6GRVP16HeCR (FIG. 37)(or pMF7GRVP16HeCR) (FIG. 38).

C. Results—Addition of Strong Activation Domains Enhance Ecdysone SwitchSystem

The VP16 transactivation domain from herpes simplex virus has been addedto a maize protoplast vector pMF6GRHEcR to generate the vectorpMF6GRVP16HEcR. When co-transformed into maize protoplasts with thereporter construct p221.9GRE6, in the presence of 100 μM RH5992,enhanced levels of expression were seen over pMF6GRHEcR. FIG. 39, showsthat RH5992 is able to induce GUS levels comparable to those observedwith the positive control (p35SCaMVGUS), moreover, a dose responseeffect is observable.

VP16 enhanced vectors (pES3 and pES4) have been generated for stabletransformation of tobacco. Following transformation transgenic progenycontaining pES3 and pES4, gave elevated GUS levels following treatmentwith RH5992, relative to comparable transgenic plants containing thenon-VP16 enhanced vectors pES1 and pES2.

An enhanced mammalian vector pcDNA3GRVP16HEcR was prepared for transienttransfection of mammalian cell lines. Elevated reporter gene activitieswere obtained relative to the effector construct (pcDNA3GRHEcR) withoutthe VP16 addition.

“Acidic” activation domains are apparently “universal” activators ineukaryotes (Ptashne (1988) Nature 335 683–689). Other suitable acidicactivation domains which have been used in fusions are the activatorregions of GAL4 itself (region I and region II; Ma and Ptashne (Cell(1987) 48, 847–853), the yeast activator GCN4 (Hope and Struhl (1986)Cell 46, 885–894) and the herpes simplex virus VP16 protein (Triezenberget al (1988) Genes Dev. 2, 718–729 and 730–742).

Other acidic and non-acidic transcriptional enhancer sequences forexample from plant fungal and mammalian species can be added to thechimeric ecdysone receptor to enhance induced levels of gene expression.

Chimeric or synthetic activation domains can be generated to enhanceinduced levels of gene expression.

EXAMPLE X Optimisation by Replacement of Heliothis Ligand Binding Domainin Chimeric Effector for that of an Ecdysone Ligand Binding Domain ofAnother Species

Mutagenesis of the ecdysone ligand binding domain results in theincreased sensitivity of the chimeric receptor for activating chemical.This can be achieved by deletions in the ligand binding domain, use oferror prone PCR (Caldwell et al., PCR Meth. Applic 2, 28–33 1992), andin vitro DNA shuffling PCR (Stemmer, Nature 370, 389–391 1994). Toenhance the efficacy of the listed techniques we have developed ascreening system for enhanced levels of induced expression (see below).

An alternative strategy to the mutation of a known ligand binding domainis to identify insect species which are particularly sensitive toecdysteroid agonists. For example Spodoptera exigua is particularlysensitive to RH5992. To investigate the role of the ecdysone receptorligand binding domain in increased sensitivity to RH5992 we haveisolated corresponding DNA sequences from of S. exigua (FIG. 40, SEQ IDNO: 6). FIG. 41, shows a protein alignment of the hinge and ligandbinding domains of the Heliothis virescens (SEQ ID NO: 5) and Spodopteraexigua (SEQ ID NO: 7) ecdysone receptors. The protein sequence betweenthe two species is well conserved.

Results—Manipulation of the Ligand Binding Domain Leads to EnhancedInduced Expression

Isolation of an ecdysone ligand binding domain from another lepidopteranspecies was carried out by using degenerate oligonucleotides and PCR offirst strand cDNA (Perkin Elmer, cDNA synthesis Kit) of the chosenspecies. The degenerate oligonucleotides at the 5′ end were HingxhoA andB and at the 3′ end ligandxA/B

HingxhoA 5′ attgctcgagaaagiccigagtgcgtigticc 3′ (SEQ ID NO: 56)                        a  t HingxhoB5′ attgctcgagaacgiccigagtgtgtigticc 3′ (SEQ ID NO: 57)                        a  c LigandxA5′ ttactcgagiacgtcccaiatctcttciaggaa 3′ (SEQ ID NO: 58)                a           c ligandxB5′ ttactcgagiacgtcccaiatctcctciaagaa 3′ (SEQ ID NO: 59)                a            t

RNA was extracted from 4th instar larvae of Spodoptera exigua sinceSpodoptera exigua appears to be more sensitive to RH5992 than Heliothis(Smagghe and Degheele, 1994). The first strand cDNA was used in PCRreactions under the following conditions 20 mM Tris-HCL (pH 8.4), 50 mMKCl, 1.5 mM MgCl₂, 200 mM dNTPs (dATP, dCTP, dGTP and dTTP) and 0.02U/ml Taq DNA polymerase and in the presence of 1 μg of each Hinge (5′3′) and Ligand (5′3′) oligonucleotides. The PCR cycling conditions were94° C. for 1 minute, 60° C. for 2 minutes, and 72° C. for 1 minute and35 cycles were carried out. A sample of the completed reaction wasfractionated in a 1% agarose (w/v) 1×TBE gel, and the resulting 900 bpfragment was subcloned into pCRII vector (Invitrogen). The resultingclone (pSKSEcR 1–10) were further characterised and sequenced.

EXAMPLE XI Manipulation of Reporter Gene Promoter Regions can ModulateChemical Induced Expression

The context of the effector response element in the reporter genepromoter can be used to modulate the basal and induced levels of geneexpression. Six copies of the glucocorticoid response element were fusedto 46 bp or 60 bp of the CaMV 35S promoter sequence. When used with theeffector construct pMF7GRHEcRS the reporter gene construct containing 46bp of the CaMV 35S promoter gave reduced basal and induced levels of GUSexpression relative to the 60 bp reporter construct (FIG. 42).

Constructs for plant transformation (pES1 and ES2) have been generatedto demonstrate the size of minimal promoter can be used to modulate thebasal and induced levels of gene expression in plants.

The number and spacing of response elements in the reporter genepromoter can be adjusted to enhance induced levels of trans-geneexpression.

The utility of a two component system (effector and reporter genecassettes) allows the spatial control of induced expression. Trans-geneexpression can be regulated in an tissue specific, organ specific ordevelopmentally controlled manner. This can be achieved by driving theeffector construct from a spatially or temporally regulated promoter.

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1. A method for controlling gene expression in a cell, the methodcomprising: (a) transforming a cell with a DNA molecule, said DNAmolecule comprising a first component which encodes an ecdysonereceptor, said receptor comprising: (i) a ligand binding domain of aHeliothis virescens ecdysone steroid receptor as shown in amino acids327–545 of SEQ ID NO: 5 or a derivative of said ligand binding domainwherein said derivative comprises one or more conservatively substitutedamino acids and wherein said derivative retains a function of saidligand binding domain; (ii) a DNA binding domain; and (iii) atransactivation domain; and a second component comprising a hormoneresponse element and a target gene; and (b) applying to the transformedcell an effective exogenous inducer wherein when the ligand bindingdomain of said first component binds the effective exogenous inducer,said DNA binding domain of said first component then binds to thehormone response element of said second component and causes expressionof said target gene.
 2. The method according to claim 1, wherein thecell is a plant cell.
 3. The method according to claim 1, wherein: (a)the transactivation domain is that of a Heliothis virescens ecdysonesteroid receptor as shown as amino acids 1–1652 of SEQ ID NO: 5 or aderivative of said transactivation domain, wherein said derivativecomprises one or more conservatively substituted amino acids and whereinsaid derivative retains a function of said transactivation domain; (b)the DNA binding domain is that of a Heliothis virescens ecdysone steroidreceptor as shown as amino acids 163–228 of SEQ ID NO: 5 or a derivativeof said DNA binding domain, wherein said derivative comprises one ormore conservatively substituted amino acids and wherein said derivativeretains a function of said DNA binding domain.
 4. The method accordingto claim 1, wherein said first component comprises a nucleic acidsequence set forth in SEQ ID NO:
 4. 5. The method according to claim 1,wherein the transactivation domain and/or the DNA binding domainconsists of a transactivation domain and/or a DNA binding domain from asteroid receptor superfamily member.
 6. The method according to claim 5,wherein the transactivation domain and/or the DNA binding domain areselected from the group consisting of: (a) a glucocorticoid receptor DNAbinding domain; (b) a glucocorticoid transactivation domain: and (c) aVP16 transactivation domain.
 7. The method according to claim 2, whereinthe target gene of the second component confers tolerance to herbicides,insects, or combinations thereof.
 8. A method according to claim 1,wherein the effective exogenous inducer is [1,2-dibenzoyl, 1-tert-butylhydrazide] or [3,5-dimethylbenzoic acid 1-1(1,1-dimethylethyl)-2(4-ethylbenzoyl) hydrazide].